Apoptosis, the morphological process leading to the controlled death of a cell, is essential for cellular homeostasis in multicellular organisms (Perl, M. et al. (2005) “APOPTOSIS,” Crit. Care Med. 33(12 Suppl):S526-S529). It provides a way of eliminating cells that are damaged or differentiated cells that are no longer necessary. For example, in the brain, only about half of the embryonic neurons generated are actually present in the adult brain; those that do not survive die via apoptosis (Snyder, S. H. (1999) “DRUGS FOR A NEW MILLENNIUM,” Philos. Trans. R. Soc. Lond. B Biol. Sci. 354:1985-1994 Snyder, 1999). However, apoptosis can be activated inappropriately; in stroke, heart disease, and neurodegenerative disorders and can result in the unnecessary loss of cells and their associated functions. Apoptosis can also be suppressed inappropriately in tumor cells and cells infected by viruses. Indeed, some viruses encode proteins capable of inhibiting the apoptotic process (Hardwick, J. M. (1998) “VIRAL INTERFERENCE WITH APOPTOSIS,” Semin. Cell Dev. Biol. 9:339-349). Further, the major treatments used to kill cancer cells, radiation and chemotherapy, function by inducing apoptosis and cells that become resistant to these treatments often do so by inhibiting the apoptotic process (Hannun, Y. A. (1997) “APOPTOSIS AND THE DILEMMA OF CANCER CHEMOTHERAPY,” Blood. 89:1845-1853). Thus, an ability to control apoptosis is of fundamental importance to the treatment of numerous diseases.
There are currently two known pathways for apoptosis: an extrinsic pathway and an intrinsic pathway. Extrinsic apoptosis signaling is mediated by the activation of so called “death receptors” which are cell surface receptors that transmit apoptotic signals after ligation with specific ligands. Death receptors belong to the tumor necrosis factor receptor (TNFR) gene superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5 (Ashkenazi, A (2002) “TARGETING DEATH AND DECOY RECEPTORS OF THE TUMOUR-NECROSIS FACTOR SUPERFAMILY,” Nat. Rev. Cancer 2(6):420-430).
The mitochondrion is known to act as a major hub for the regulation of intrinsic apoptosis (Chen, H. et al. (2005) “EMERGING FUNCTIONS OF MAMMALIAN MITOCHONDRIAL FUSION AND FISSION,” Hum Mol Genet. 14 Spec No. 2:R283—R289; Mohamad, N. et al. (2005) “MITOCHONDRIAL APOPTOTIC PATHWAYS,” Biocell. 29(2):149-61). Early in mitochondria-mediated apoptosis, there is an increase in the permeability of the mitochondrial outer membrane (MOM) that leads to the release of intermembrane space proteins, including cytochrome c, procaspases, apoptosis-inducing factor (AIF), heat shock proteins, Smac/Diablo, and endonuclease G (Saelens, X. et al. (2004) “TOXIC PROTEINS RELEASED FROM MITOCHONDRIA IN CELL DEATH,” Oncogene 23:2861-2874). The release of these proapoptotic proteins into the cytoplasm is crucial for the activation of specific caspases and DNases that are responsible for the execution of apoptosis. The key event of this mitochondrial pathway is the permeabilization of the mitochondrial outer membrane (MOM) to proteins and the efflux of apoptosis-inducing intermembrane space proteins such as cytochrome c into the cytoplasm. The release of these proteins leads to the activation of the caspases that carry out the execution phase of apoptosis (Crompton, M. (1999) “THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE AND ITS ROLE IN CELL DEATH,” Biochem. J. 341:233-249; Susin, S. A. et al. (1998) “MITOCHONDRIA AS REGULATORS OF APOPTOSIS: DOUBT NO MORE,” Biochim. Biophys. Acta. 1366:151-165). Cells can be rescued before this step but are generally fated to die once the proapoptotic proteins have been released from mitochondria. Therefore, an understanding of the mechanism for the permeabilization of the MOM to proteins is relevant to developing methods for controlling apoptosis.
A number of mechanisms have been proposed for the protein permeation pathway. Candidates for the composition of this pore include Bax oligomers (Antonsson, B. et al. (2000) “BAX OLIGOMERIZATION IS REQUIRED FOR CHANNEL-FORMING ACTIVITY IN LIPOSOMES AND TO TRIGGER CYTOCHROME C RELEASE FROM MITOCHONDRIA,” Biochem. J. 345:271-278; Antonsson, B. (2001) “BAX IS PRESENT AS A HIGH MOLECULAR WEIGHT OLIGOMER/COMPLEX IN THE MITOCHONDRIAL MEMBRANE OF APOPTOTIC CELLS,” J. Biol. Chem. 276:11615-11623; Saito, M. et al. (2000) “BAX-DEPENDENT TRANSPORT OF CYTOCHROME C RECONSTITUTED IN PURE LIPOSOMES,” Nat. Cell Biol. 2:553-555; Pavlov, E. V. et al. (2001) “A NOVEL, HIGH CONDUCTANCE CHANNEL OF MITOCHONDRIA LINKED TO APOPTOSIS IN MAMMALIAN CELLS AND BAX EXPRESSION IN YEAST,” J. Cell Biol. 155:725-73), the mitochondrial apoptosis-induced channel MAC (Pavlov, E. V. et al. (2001) “A NOVEL, HIGH CONDUCTANCE CHANNEL OF MITOCHONDRIA LINKED TO APOPTOSIS IN MAMMALIAN CELLS AND BAX EXPRESSION IN YEAST,” J. Cell Biol. 155:725-731), lipidic pores induced by Bax (Basañez, G. et al. (1999) “BAX, BUT NOT BCL-XL, DECREASES THE LIFETIME OF PLANAR PHOSPHOLIPID BILAYER MEMBRANES AT SUBNANOMOLAR CONCENTRATIONS,” Proc. Natl. Acad. Sci. USA 96:5492-5497), lipidic pores induced by BH3/Bax/lipid interactions (Kuwana, T. et al. (2002) “BID, BAX, AND LIPIDS COOPERATE TO FORM SUPRAMOLECULAR OPENINGS IN THE OUTER MITOCHONDRIAL MEMBRANE,” Cell 111:331-342; Terrones, O. et al. (2004) “LIPIDIC PORE FORMATION BY THE CONCERTED ACTION OF PROAPOPTOTIC BAX AND TBID,” J. Biol. Chem. 279:30081-30091), interactions between Bax and ceramide (Pastorino, J. G. et al. (1999) “FUNCTIONAL CONSEQUENCES OF THE SUSTAINED OR TRANSIENT ACTIVATION BY BAX OF THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE,” J. Biol. Chem. 274:31734-31739), and ceramide channels (Siskind, L. J. et al. (2000) “THE LIPIDS C2-AND C16-CERAMIDE FORM LARGE STABLE CHANNELS. IMPLICATIONS FOR APOPTOSIS,” J. Biol. Chem. 275:38640-38644; Siskind, L. J. et al. (2002) “CERAMIDE CHANNELS INCREASE THE PERMEABILITY OF THE MITOCHONDRIAL OUTER MEMBRANE TO SMALL PROTEINS,” J. Biol. Chem. 277:26796-26803; Siskind, L. J. et al. (2003) “ENLARGEMENT AND CONTRACTURE OF C2-CERAMIDE CHANNELS,” Biophys. J. 85:1560-1575).
A variety of assays have been developed to assess apoptosis. For example, apoptosis has been assayed using DNA stains able to detect the breakdown of the cell nucleus (see, for example, U.S. Pat. No. 6,248,904; Briant, L. et al. (1998) “INVOLVEMENT OF EXTRACELLULAR SIGNAL-REGULATED KINASE MODULE IN HIV-MEDIATED CD4 SIGNALS CONTROLLING ACTIVATION OF NUCLEAR FACTOR-KAPPA B AND AP-1 TRANSCRIPTION FACTORS,” J. Immunol. 160:1875-1885); by DNA electrophoresis or flow cytometry (see, for example, Gong, J, et al. (1994) “A SELECTIVE PROCEDURE FOR DNA EXTRACTION FROM APOPTOTIC CELLS APPLICABLE FOR GEL ELECTROPHORESIS AND FLOW CYTOMETRY,” Anal. Biochem. 218:314-319; Belloc, F. et al. (1994) “A FLOW CYTOMETRIC METHOD USING HOECHST 33342 AND PROPIDIUM IODIDE FOR SIMULTANEOUS CELL CYCLE ANALYSIS AND APOPTOSIS DETERMINATION IN UNFIXED CELLS,” Cytometry 17:59-65; Singh, N. P. et al. (1994) “MODIFICATIONS OF ALKALINE MICROGEL ELECTROPHORESIS FOR SENSITIVE DETECTION OF DNA DAMAGE,” Int. J. Radiat. Biol. 66:23-28 (1994). Apoptosis has been assayed using caspase assays (Darzynkiewicz, Z. et al. (2001) “FLOW CYTOMETRY IN ANALYSIS OF CELL CYCLE AND APOPTOSIS,” Semin Hematol. 38(2):179-93); Riss, T. L. (2001) “APOPTOSIS AS A BIOMARKER IN CHEMOPREVENTION TRIALS,” Urology. 57(4 Suppl 1): 141-142; Saraste, A. et al. (2000) “MORPHOLOGIC AND BIOCHEMICAL HALLMARKS OF APOPTOSIS,” Cardiovasc Res. 45(3):528-537). Various assays have been developed to detect apoptosis based on changes that occur in the permeability of cell membranes (see, for example, Idziorek, T. et al. (1995) (“YOPRO-1 PERMITS CYTOFLUOROMETRIC ANALYSIS OF PROGRAMMED CELL DEATH (APOPTOSIS) WITHOUT INTERFERING WITH CELL VIABILITY,” J. Immunol. Methods 185:249-258; Susin, S. A. et al. (1998) “MITOCHONDRIA AS REGULATORS OF APOPTOSIS: DOUBT NO MORE,” Biochim. Biophys. Acta 1366:151-165; Green, D. R. et al. (1998) “MITOCHONDRIA AND APOPTOSIS,” Science 281:1309-1312).
Despite all such progress, a need remains for improved methods for assaying apoptosis and for identifying compounds that affect apoptosis. The identification of such compounds would provide an ability to control apoptosis and thereby permit the treatment of major diseases such as cancer, stroke, heart disease, neurodegenerative diseases, and viral diseases, for which current treatments are seriously inadequate. The present invention is directed to these and other needs.